The DNA mismatch repair (MMR) pathway performs a critical role in the maintainence of genomic stability. Defects in this pathway lead to the destabilization of microsatellite tracts and have been correlated with the cancer syndrome hereditary nonpolyposis colorectal cancer (HNPCC), sporadic tumorigenesis, and resistance to various chemotherapeutics. To date, the majority of HNPCC cases have been correlated with mutations in four MMR genes, hMSH2, hMLH1, hPMS2, and hPMS1 that account for approximately 70% of the known HNPCC families. In S. cerevisiae and humans, the MutS Homologs MSH2, MSH3 and MSH6 are responsible for the recognition of DNA mispairs. However, the steps following lesion binding are unclear. The human exonuclease 1 (hEXO1) protein interacts with hMSH2, hMLH1 and hMSH3 and is involved in the removal of DNA mispairs. In order to further our understanding of the human mismatch repair process, we propose to characterize the activity of hEXO1 in conjunction with the hMSH2-hMSH3, hMSH2-hMSH6, and hMLHI-hPMS2 MMR complexes and to elucidate the role of hEXO1 acetylation in mismatch repair. This will be accomplished in two specific aims. In specific aim 1, we will test the hypothesis that the proteins involved in the early steps of mismatch repair modulate the activity of hEXO1 by examining the cooperation between hEXO1 and hMSH2-hMSH3, hMSH2-hMSH6, and hMLHI-hPMS2 in the removal of DNA mismatches and insertion/deletion loops. In specific aim 2, we test the hypothesis that acetylation of hEXO1 modulates the MMR activities of this protein. We will examine the effect of acetylation on the nuclease, DNA binding, and protein-protein interaction capabilities of hEXO1.